Functional genomics often requires the construction of gene delivery systems to force the expression of recombinant cDNA sequences in live cells. The resulting specific protein product can then be studied for its cellular localization and influence on cellular function, differentiation and proliferation, as well as a host of phenotypic changes. The state of the art is such that any cDNA sequence of interest can be delivered to the cell and become transiently or permanently part of the cellular genome by the use of modified animal viruses. Murine retroviral vectors have emerged in the past several years as the most common vehicle to deliver marker genes. Other viral vectors such as lentiviruses, adenoviruses, herpes viruses, adeno-associated viruses, and non-viral methods such as plasmids have also been used for gene transfer.
The efficiency of gene delivery systems varies according to the gene delivery vector and the target cell type to which the genes are transferred. Thus, the recombinant DNA molecule used in gene delivery systems requires the incorporation of additional polynucleotide sequences which mediate the expression of a second protein product, which confers different phenotypic properties to genetically modified cells. These altered phenotypic characters allow for positive or negative selection of cells in different kinds of assays. In a positive selection assay, this genetic marker is used to confirm the successful introduction of the recombinant DNA molecule into the cell, and is also important in the isolation of an exclusive population of cells which expresses the recombinant DNA molecule of interest. All cells that do not express the marker are excluded from the population of interest. This allows for an accurate study of the effects of primary cDNA sequence on cellular function and phenotype.
The marker usually encodes enzymes that confer antibiotic resistance, or enzymes that complement a metabolic deficiency, fluorescent proteins, or surface markers that can be selected using appropriate ligands. Gene transfer systems also include markers such as β-galactosidase, luciferase, and alkaline phosphatase. Detection of these markers involves either cell fixation that kills the cells and the addition of a substrate. These methods are often time consuming and are prone to endogenous high background. Another group of gene transfer markers convey drug resistance and thus allow positive selection of transfected cells through selection of resistant colonies. Although drug selectable markers allow the detection of living cells by expressing the transgene that encodes an enzyme that modifies the antibiotic molecule, they require that the cells survive in a toxic environment over a long period of time. The consequences of selection methods which use antibiotics include issues of toxicity and effects on normal gene expression profiles which, in turn, can affect the interpretation of phenotypic changes associated with the introduction of recombinant DNA molecules. In addition, long selection periods associated with antibiotics can severely limit the window of time available to researchers for functional genomic analysis or cell biological observations regarding genes of interest where cells senesce takes place in culture within a short period of time. For example, the neomycin-resistance gene, which confers resistance to the neomycin analog G418, has been shown to have deleterious effects upon the expression of other genes in retroviral vectors. (Emerman, M., et al. (1986) Nucleic Acids Res. 14, 9381–9396).
More recent advances in gene delivery systems utilize a sortable marker encoded by recombinant cDNA sequences to result in a protein product that is capable of fluorescence within the cell (GFP, for example). These systems have the advantage over selection markers in that isolation of a cell population of interest can often be more rapid than with antibiotics. The disadvantage of these fluorescent, sortable markers is that they can suffer from lack of sensitivity. Furthermore, in certain cells, the level of internal auto fluorescence from a cell can overlap and interfere with the true fluorescent signal of the marker, thus preventing the efficient isolation of cells that have been modified by the delivery of recombinant DNA molecules.
Other types of selectable markers that allow for cell sorting of transduced or transfected cells are proteins that introduce epitopes on the extracellular side of the cell membrane. These membrane proteins are detected with specific ligands or antibodies that bind to extracellular domains of these proteins. This allows for fluorescence activated cell sorting if the antibody or ligand are directly or indirectly labeled with fluorescent molecules, or for magnetic sorting if the antibody or ligand are directly or indirectly linked to ferromagnetic beads. This strategy has the advantage of being highly flexible as it allows different cell sorting methods and analysis of transduced cells, but suffers from the disadvantage that it is not be very sensitive. Also, expression of transmembrane proteins might alter the phenotypic characteristics of the cell under analysis.
Controlling the expression of the introduced recombinant DNA molecule such that it can be limited to a phenotypically-distinct subset of cells within a larger general population provides additional advantages to the live isolation of specific cell types where presently no or few adequate cell-surface markers exist. Epithelial adult stem cells provide one example where few, if any, adequate cell surface markers allow for the isolation of the stem cell population in most epithelial organs upon disaggregation of the epithelial tissue. However, there exist a number of cytoplasmic and nuclear proteins which do readily mark the epithelial stem cell population within an epithelial tissue. Thus, the introduction of a phenotypic-specific selectable marker to drive the expression of cell surface epitopes would allow for the isolation of a distinct subpopulation of modified cells that adhered to the phenotype of an adult stem cell of a given epithelial organ. Similarly, there are also few identified unique cell surface markers to adequately separate tumor cells from normal epithelia within an epithelial tissue. This inability to actively sort phenotypically distinct cells hampers clinical and diagnostic research efforts in cancer biology. Correctly identifying and isolating the slow growing tumor cell population from the rest of the epithelial tissue could therefore yield greater progress in identifying those genes which are truly altered in mechanisms of cancer. Thus, the invention of an innocuous marker with no discernable toxicity that is strongly detectable and can enable the rapid isolation of a population of marked cells would offer great advantages to the fields of finctional genomics and cell biology.
It is therefore a primary objective of the present invention to provide a gene transfer marker that overcomes the deficiencies of currently available gene transfer markers as described above.
It is another objective of the present invention to provide a gene transfer marker that provides rapid and sensitive identification of gene transfer in living mammalian cells.
These and other objectives will become apparent from the following description.